Cloning of Cutinase Transcription Factor 1, a Transactivating Protein Containing Cys6Zn2 Binuclear Cluster DNA-binding Motif

Abstract

Hydroxy fatty acids from plant cutin were shown previously to induce the expression of the cutinase gene via a palindromic sequence located at ؊159 base pairs of the cutinase gene in Fusarium solani f. sp. pisi (Nectria hematococca mating type VI). Of the two overlapping palindromes in this sequence, palindrome 2 was found to be essential for the inducibility of cutinase by hydroxy fatty acids. Screening of a phage expression library with the concatenated palindrome 2 as probe detected a distinct cDNA clone encoding a polypeptide designated cutinase transcription factor 1␣ (CTF1␣) with a calculated molecular weight of 101,109. This protein contains a Cys 6 Zn 2 binuclear cluster motif sharing homology to the Cys 6 Zn 2 binuclear cluster DNA-binding domains of transcription factors from Saccharomyces cerevisiae, S. carlsbergensis, Kluyveromyces lactis, Neurospora crassa, Aspergillus nidulans, and A. flavus. CTF1␣, expressed in Escherichia coli, showed specific binding to the palindrome 2 DNA fragment but not to palindrome 1 or mutant palindrome 2 DNA fragments, suggesting specific binding of CTF1␣ to palindrome 2. When CTF1␣ was expressed as a fusion protein with the nuclear localization sequence of SV40 in yeast, it transactivated the native cutinase promoter fused to the chloramphenicol acetyl transferase (cat) gene. Mutation of palindrome 2 but not palindrome 1 abolished this transactivation. Thus, CTF1␣ positively acts in vivo by binding selectively to palindrome 2 of the cutinase gene promoter.

Regulation of Constitutively Expressed and Induced Cutinase Genes by Different Zinc Finger Transcription Factors inFusarium solani f. sp. pisi(Nectria haematococca)

“…The transactivation was measured by the level of CAT activity. Such an approach has been used previously to demonstrate transactivation of cut1 promoter in vivo (7). With this approach, CTF1␤ was found to activate the native cut1 promoter only slightly (Fig.…”

“…Phage DNA purified from these clones was double digested with EcoRI and BamHI, EcoRI and SalI, EcoRI and SstI, or EcoRI and XhoI. The phage clones whose DNA differed in restriction patterns were probed with 32 P-labeled palindrome 2 fragment as described (7). Two polypeptides from two of the phage clones, designated ctf15 and ctf11, that bound the concatenated palindrome 2 were designated CTF1␣ (7) and CTF1␤, respectively.…”

“…Yeast transformants were selected on leucine-and histidine-lacking minimal medium (7). Growth of yeast transformants and CAT assays were done as described (7).…”

Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca

“…These products would then activate the transcription of the inducible gene, generating a high level of the enzyme that can assist the pathogen to penetrate through the physical barriers of the host. This concept has been demonstrated for cutinase, and the transcription factors involved in the expression of the induced gene (48,49) and the constitutively expressed gene (T. Sirakova, D. Li, and P.E.K., unpublished work) have been cloned from N. hematococca. In this fungus, the constitutively expressed gene, pelB, was suggested to provide the oligomers that induce pelA, because antisense expression of pelB prevented induction of pelA (W. Guo and P.E.K., unpublished work).…”

A GAL4-like Protein Is Involved in the Switch between Biotrophic and Necrotrophic Phases of the Infection Process of Colletotrichum lindemuthianum on Common Bean

“…The deduced amino acid sequence of the CLTA1 protein suggests that it contains the three functional domains characteristic of the GAL4-like family of transcriptional activators (Schjerling and Holmberg, 1996): the C 6 zinc cluster involved in DNA binding, an MHR, and a possible transcriptional activation domain. In Fusarium solani f sp pisi, another Zn(II) 2 Cys 6 protein, CTF1, involved in transcriptional activation of a cutinase-encoding gene, was identified (Li and Kolattukudy, 1997). However, gene replacement experiments still need to be performed to demonstrate its role in fungal pathogenicity.…”